Volume 59, Issue 1, Pages 137-146 (January 2001) Clusterin is up-regulated in glomerular mesangial cells in complement-mediated injury  Koei Yamada, Yuichi Hori, Norio Hanafusa, Toshihiro Okuda, Nobuo Nagano, Nam-Ho Choi-Miura, William G. Couser, Toshio Miyata, Kiyoshi Kurokawa, Toshiro Fujita, Masaomi Nangaku  Kidney International  Volume 59, Issue 1, Pages 137-146 (January 2001) DOI: 10.1046/j.1523-1755.2001.00474.x Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 1 Temporal profile of secretion of clusterin protein by rat mesangial cells stimulated by sublytic complement attack. (A) Western blot of concentrated culture medium of rat mesangial cells. Each lane contains 100 μg of protein. Clusterin is present as an α-subunit of 35 kD under reducing conditions. The data are representative of four separate experiments. (B) Densitometric analysis of each lane. Data are expressed as percentage of control (0 hour, □) of four separate experiments. The peak of clusterin in the cultured media is at 24 hours after the stimulation (*P < 0.05). (C) A time control for the time course of the expression of clusterin protein. The expression of clusterin in rat mesangial cells without complement attack did not increase over time. The data are representative of four separate experiments. Kidney International 2001 59, 137-146DOI: (10.1046/j.1523-1755.2001.00474.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 2 Western blot of clusterin secreted by rat mesangial cells stimulated by sublytic complement attack. (A) Concentrated supernatant protein (100 μg) was separated by SDS-PAGE under reducing conditions and was processed for immunoblotting with anticlusterin antibody as described in the Methods section. The data are representative of four separate experiments. (B) Densitometric analysis of four separate experiments. Data are represented as percentage of control (□) for each experiment. Clusterin was markedly increased in the supernatants of mesangial cells at 24 hours after stimulated by sublytic complement attack. Either ATS IgG, 5% rat serum, or DMEM alone did not induce up-regulation of clusterin (*P < 0.05). (C) Western blot of the culture medium of rat mesangial cells incubated with 5% C6-sufficient rat serum or 5% C6-deficient rat serum after ATS sensitization. No increased expression of clusterin was seen in rat mesangial cells at 24 hours after the incubation in 5% C6-deficient rat serum with ATS sensitization. The data are representative of four separate experiments. Kidney International 2001 59, 137-146DOI: (10.1046/j.1523-1755.2001.00474.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 3 Induction of clusterin mRNA in cultured rat mesangial cells stimulated by sublytic complement attack. (A) Northern blot for total RNA (10 μg per lane) from rat mesangial cells with or without stimulation by C5b-9 attack was probed for clusterin and GAPDH. The data are representative of four separate experiments. (B) Densitometric quantitation of the blots. The results were expressed as means ± SD (N = 4) of the relative ratio of clusterin/GAPDH mRNA. The expression of clusterin mRNA was significantly increased in rat mesangial cells at 24 hours after the incubation of 5% normal rat serum with ATS sensitization compared with that of DMEM alone (*P < 0.05). Kidney International 2001 59, 137-146DOI: (10.1046/j.1523-1755.2001.00474.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 4 Induction of clusterin mRNA in anti-Thy1 nephritis. (A) Northern blot of glomerular total RNA (10 μg per lane) extracted from control rats 8 days (N = 6) and 29 days (N = 6) after vehicle injection, and experimental rats 8 days (N = 6) and 29 days (N = 6) after OX-7 injection. Each lane represents a separate rat. The Northern blot was hybridized with a clusterin cDNA probe and a GAPDH cDNA probe. (B) Densitometric quantitation of the blots. The results are expressed as means ± SD (N = 6) of the clusterin/GAPDH mRNA ratio. A marked induction of clusterin mRNA is seen in the anti-Thy1 nephritis rats compared with control rats (*P < 0.05). Kidney International 2001 59, 137-146DOI: (10.1046/j.1523-1755.2001.00474.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 5 In situ hybridization for clusterin mRNA in the glomeruli of the OX-7–treated rats (A and B) and control rats (C and D) on day 8 using antisense (A and C) and sense (B and D) probes. Note the increased expression of clusterin mRNA in the glomeruli of the OX-7–treated rats (A) compared with control rats (C; ×400). No signal is observed in clusterin sense-probed kidney sections (B and D; ×400). Kidney International 2001 59, 137-146DOI: (10.1046/j.1523-1755.2001.00474.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 6 Immunohistochemical analysis shows increased deposition of clusterin in anti-Thy1 nephritis rats. Clusterin staining of the OX-7–treated rats (A and C) or control rats (B and D) at day 8 (A and B) and at day 29 (C and D). Significantly increased staining of clusterin was seen in the glomeruli of the OX-7–treated rats (×400). Kidney International 2001 59, 137-146DOI: (10.1046/j.1523-1755.2001.00474.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 7 Semiquantitation of clusterin staining confirmed increased deposition of clusterin. Clusterin staining in the glomeruli was significantly increased in the OX-7–treated rats both at day 8 and at day 29 compared with control rats. *P < 0.05. Kidney International 2001 59, 137-146DOI: (10.1046/j.1523-1755.2001.00474.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 8 Clusterin colocalized with C5b-9 in glomeruli of anti-Thy1 nephritis rats on day 8. Double immunofluorescent staining for clusterin in red (Texas red X; A) and C5b-9 in green (Oregon green; B) demonstrates the overlapping distribution of these proteins during complement-mediated injury. The overlapping area appeared orange on the merged images (C; ×400). Kidney International 2001 59, 137-146DOI: (10.1046/j.1523-1755.2001.00474.x) Copyright © 2001 International Society of Nephrology Terms and Conditions